I would like to thank Michael Caravito and Gert Van den Berg for
taking the time to share their knowledge and insights on
protein-detergent/micelle complexes.
A series of experiments I carried out using DLS some time ago showed
that the protein-detergent/micelle complex (with LDAO as detergent)
for the membrane protein I was studying had a hydrodynamic radius that
was 20 angs larger than the empty-micelles. The experiments were done
in the protein storage buffer: 20 mM Tris 8.0, 250 mM NaCl, 0.15 %
LDAO, which is far from a crystallization condition!
As Michael and Gert pointed out, it is a risky business to
draw quantitative conclusions from such measurements due to the
effects of so many factors. Nonetheless, it was quite
informative to me at the time to see a marked difference between the
protein-detergent complex and the empty-micelles. Aside form bringing
home the concept that the interaction of the protein with
detergent/micelle had a true physical meaning that could be translated
to particle augmentation, it also helped me benchmark my
gel-filtration runs.
Best regards
Savvas
----
Savvas N. Savvides
Unit for Structural Biology and Biophysics
Laboratory for Protein Biochemistry - Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19
Email: [log in to unmask]
http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html
Quoting "R.M. Garavito" <[log in to unmask]>:
> Saavas and Tommi,
>
> The questions of what is the detergent content of a membrane protein
> crystal and how to explicitly determine the amount of detergent in a
> crystal are extremely difficult to address. Moreover, is it
> worthwhile to even attempt to correct the Matthews coefficient? I
> personally don't for a number of reasons. However, one point I would
> like to make in this discussion is that ANYTHING concerning micellar
> structure or behavior cannot be naively extrapolated to a protein-
> detergent complex without firm experimental data. Moreover, when the
> protein-detergent complex is in a crystal, it gets even worse. Very
> little quantitative work has been done on what is the detergent
> structure and behavior in a protein-detergent complex. Peter Timmins
> has done the most using neutron diffraction with me and Wolfram Welte
> on crystalline systems, as well as in solution (one paper is below).
>
> Pebay-Peyuola, E., Garavito, R.M., Rosenbusch, J.P., Zulauf, M., and
> Timmins, P.A. (1995) "Detergent structure in tetragonal crystals of
> porin from the outer membrane of E. coli." Structure 3, 1051-1059.
>
> One immediate take home message is that a membrane protein IS NOT in a
> micelle, even by definition from surfactant chemistry, nor does a
> membrane protein insert into a micelle. In many of the experiments on
> detergent binding in surfactant chemistry using styrene beads,
> detergent adsorbs onto a hydrophobic surface from single monomer
> accretion, and perhaps by micelle fusion. Hence, one should forget
> about micelles when talking about a protein-detergent complex. My
> rule of thumb from experience is that an "average" membrane protein of
> about 50 KD binds about a micelle's worth of detergent, but it would
> be a mistake to assume it has all the characteristics of a free and
> pure detergent micelle.
>
> Getting back to the amount of detergent in a crystal and the Matthews
> coefficient, the detergent layer of protein-detergent complex can
> behave like a hard sphere in a crystal or it can fuse with its
> neighbors, depending on the detergent used. Changing the detergent
> concentration around the crystal, as we do when manipulating a crystal
> for many experiments, will change the detergent concentration in the
> crystal and can impact the detergent layer of protein- detergent
> complex. Thus, efforts to get accurate, detergent- corrected Matthews
> coefficients for membrane proteins, may not be worth worrying about.
>
> Regards,
>
> Michael
>
> ****************************************************************
> R. Michael Garavito, Ph.D.
> Professor of Biochemistry & Molecular Biology
> 513 Biochemistry Bldg.
> Michigan State University
> East Lansing, MI 48824-1319
> Office: (517) 355-9724 Lab: (517) 353-9125
> FAX: (517) 353-9334 Email: [log in to unmask]
> ****************************************************************
>
>
> On Sep 24, 2007, at 2:29 AM, Tommi Kajander wrote:
>
>> Quoting Edward Berry <[log in to unmask]>:
>>
>>> Savvas Savvides wrote:
>>>> Indeed, but wouldn't consideration of micelle size affect our
>>>> estimation of the number of molecules in the asu, in some cases
>>>> significantly?
>>> Good point- I think now that is taken into account by just saying
>>> "membrane proteins tend to have a high solvent content" and taking
>>> that into consideration when you guess the number of molecules.
>>> But it would be nice to account for the detergent explicitly.
>>> Say by analyzing detergent content of the crystals, or in some
>>> ideal cases neutron diffraction with perdeuterated detergent.
>>>
>>
>>
>> with regard to this has anyone actually checked how the micelle properties
>> with or without protein "embedded" might differ?? are we assuming empty
>> micelle and the protein-added micelle are the same size/Mw? is this really
>> so?
>> --- of course this may further vary depending on the oligomeric
>> state of the
>> protein --suppose some neutron scattering studies on model systems might
>> give the answer --havent looked. just wondering..
>>
>> -tommi
>>
>>
>>
>>
>> --
>> Tommi Kajander, Ph.D.
>> Macromolecular X-ray Crystallography
>> Research Program in Structural Biology and Biophysics
>> Institute of Biotechnology
>> PO box 65 (Street address: Viikinkaari 1, 4th floor)
>> University of Helsinki
>> FIN-00014 Helsinki, Finland
>> Tel. +358-9-191 58903
>> Fax +358-9-191 59940
>>
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